Seasonal Shifts in Bacterial and Fungal Microbiomes of Leaves and Associated Leaf-Mining Larvae Reveal Persistence of Core Taxa Regardless of Diet

ABSTRACT Microorganisms are key mediators of interactions between insect herbivores and their host plants. Despite a substantial interest in studying various aspects of these interactions, temporal variations in microbiomes of woody plants and their consumers remain understudied. In this study, we investigated shifts in the microbiomes of leaf-mining larvae (Insecta: Lepidoptera) and their host trees over one growing season in a deciduous temperate forest. We used 16S and ITS2 rRNA gene metabarcoding to profile the bacterial and fungal microbiomes of leaves and larvae. We found pronounced shifts in the leaf and larval microbiota composition and richness as the season progressed, and bacteria and fungi showed consistent patterns. The quantitative similarity between leaf and larval microbiota was very low for bacteria (~9%) and decreased throughout the season, whereas fungal similarity increased and was relatively high (~27%). In both leaves and larvae, seasonality, along with host taxonomy, was the most important factor shaping microbial communities. We identified frequently occurring microbial taxa with significant seasonal trends, including those more prevalent in larvae (Streptococcus, Candida sake, Debaryomyces prosopidis, and Neoascochyta europaea), more prevalent in leaves (Erwinia, Seimatosporium quercinum, Curvibasidium cygneicollum, Curtobacterium, Ceramothyrium carniolicum, and Mycosphaerelloides madeirae), and frequent in both leaves and larvae (bacterial strain P3OB-42, Methylobacterium/Methylorubrum, Bacillus, Acinetobacter, Cutibacterium, and Botrytis cinerea). Our results highlight the importance of considering seasonality when studying the interactions between plants, herbivorous insects, and their respective microbiomes, and illustrate a range of microbial taxa persistent in larvae, regardless of their occurrence in the diet. IMPORTANCE Leaf miners are endophagous insect herbivores that feed on plant tissues and develop and live enclosed between the epidermis layers of a single leaf for their entire life cycle. Such close association is a precondition for the evolution of more intimate host-microbe relationships than those found in free-feeding herbivores. Simultaneous comparison of bacterial and fungal microbiomes of leaves and their tightly linked consumers over time represents an interesting study system that could fundamentally contribute to the ongoing debate on the microbial residence of insect gut. Furthermore, leaf miners are ideal model organisms for interpreting the ecological and evolutionary roles of microbiota in host plant specialization. In this study, the larvae harbored specific microbial communities consisting of core microbiome members. Observed patterns suggest that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than generally presumed.

This study investigated seasonal shifts in the microbiomes of leaf mining larvae (Lepidoptera and Hymenoptera) and their host trees over one growing season in a deciduous temperate forest. The results not only highlight the importance of considering seasonality in the studies of the interactions between plants, herbivorous insects, and their respective microbiomes, but also clarified that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than previously presumed. This is potentially an important contribution to the insect microbiome research field; however, there are several concerns that need to be addressed.
The authors collected both Lepidoptera and Hymenoptera insects, and since the biology of the two insect orders are too different, I would suggest to separate them in the analysis and then compare the two insect orders. Furthermore, is there any interspecies differences? Whether some insect hosts have more specific gut microbiota or not.
L343,"the changes were not significant", the statistical analyses (test method, P value...) should be clearly indicated in the related figure caption. Most of comparisons in the figures need be clarified.
High throughput sequencing is semi quantitative, representing only relative abundances. Validating some of the core taxa identified in this study (at least by Quantitative PCR) will provide more convincing evidence for the main conclusion here, because there is strong debate on the microbial residence of lepidopteran gut.
A table summarizing the taxa prevalent in larvae, in leaves and in both leaves and larvae as well as their potential functions (in the discussion) would provide more clear information.
Minors: 1. Table S5 Existing variables are less explanatory for microbial composition, and more variables should be introduced, such as temperature. 2. Table S5 Why not distinguish the sampling sites of insects, like trees (Sampling plot)? Even if the same insect, its microbiome may also vary depending on the sampling plot (see: Foliar-feeding insects acquire microbiomes from the soil rather than the host plant). 3. State the main results more clearly by adding relevant statistics to the graphs and legends. This is the basic requirement for figures and legends. 4. Fig. 8, Why choose frequency instead of showing the average relative abundance of the Top ten microorganisms? 5. L297-L299, relative abundances are useful information, microbiota composition of leaf and larvae throughout the season should be described. 6. L283-L289, please provide a complete table of statistical results. 7. L18, L89 "ITS2 rRNA metabarcoding", rRNA gene or rDNA like 16S rDNA, and why select this set of primer for fungi (ITS2 region)? 8. L392, wrong citations, the reference 84 studies Enterococcus mundtii but not Streptococcus, please check.
Reviewer #2 (Comments for the Author): The authors describe the microbiota of insects and their leaf environments from a deep sample pool to address questions about the importance of the lepidopteran microbiota. The data are interesting given ongoing debate about the importance of the lepidopteran microbiota. In my opinion the data are beautifully and rigorously presented. The paper was a pleasure to read and the methods robust and informative.
However, I think there is an issue the authors should address to make their results clearer. The authors include a few Tenthredinidae in their analysis, but I think this might detract from the overall message. Does the message change if these few samples are excluded, so that the text's comparisons with other lepidopteran work are not confounded? If so, then this could drive the authors' detection of patterns that are at least somewhat in contrast to the work exemplified by Hammer. If not, I see no tangible benefit to retaining a few insect samples that otherwise convolute the analysis. I recommend the authors perform a reanalysis and drop the samples; or explain why they should be retained (I may be missing something important).
Some readers may wish to see quantitative estimates of bacterial abundance, as others have argued that this is an objective metric that can determine the 'true' microbiota. I'm not in this camp, but the authors may wish to proactively include some qPCR data. I think the data may not be necessary, because the authors comparisons, e.g. using the Renkonen index, are mostly independent of these concerns (the patterns emerge whether abundant or not). See also my comment abount neutral models below, too, which also applies to patterns. But, readers will likely be interested in it and it would lend strength to the author's points.
I have several small suggestions: L123 -form > from Please provide brief methods details at L123 (Hrcek) and L130 (ref 48). L135 -please describe the minor modification L170 -were any archaeal reads detected and, if so, were they discarded? L238 -please explain where the 12.8% etc values and the 7.87% etc values come from -are these a separate permanova comparing April to other time points, and then comparing within June, august, and October? Please clarify. Same question arises at L245-7 L288 -please edit this sentence for clarity (twice less?) L320 -would 'relatively' be better than 'very'? L326 -with > "by also describing"? L332 -alters > changes L362 -these numbers vary slightly from the table -please synchronize them. L440 -the authors allude to neutral modeling; I think using the sncm_fit method of sloan 2006 as implemented in burns 2016 (https://www.nature.com/articles/ismej2015142) would be trivial, and could provide an additional layer of insight. I know burnout on additional experiments is real, but my lab recently tried it and in only takes a few minutes to get the script running. I'd be happy to provide some guidance or links if the authors are interested to try it.

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Thank you for submitting your paper to Microbiology Spectrum.

November 2022
Dr John Chaston Editor, Microbiology Spectrum Dear Dr Chaston, Thank you for considering our manuscript entitled "Seasonal shifts in bacterial and fungal microbiomes of leaves and associated leaf-mining larvae reveal persistence of core taxa regardless of diet" (Spectrum03160-22) for publication. I, along with my co-authors, would like to re-submit its revised version.
We received two positive reviews on our manuscript. We carefully checked the manuscript and made appropriate changes in accordance with the reviewers' suggestions. The most extensive change to emerge from the reviews was the omission of hymenopteran samples from the analyses. However, the results and the main significance remained unchanged. We also decided not to include qPCR analysis in our study (see the reasoning below). We believe that the inspiring and relevant comments of both reviewers have significantly improved the overall quality of our manuscript. Our responses are attached herewith.
We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have. Sincerely,

Hana Šigutová
Department of Biology and Ecology University of Ostrava, Czech Republic hana.sigutova@osu.cz

Editor
Thank you for submitting your manuscript to Microbiology Spectrum. I apologize for the delay, but one reviewer was unable to complete the review after initially accepting, and it added an unusual extension to the normal review time. Your manuscript has now been reviewed by two experts in the field, and you can see that both reviewers viewed your manuscript positively. Each raised some concerns, however, and I agree with these reviews. The reviewers are split on the importance of a qPCR analysis, and I would emphasize two lines of guidance: 1) if you choose to do the qPCR, it could be on a subset of the many samples you analyzed; 2) if you choose not to do the analysis, you should justify your reasoning sufficiently in both the text and reviewer response to address both reviewer's points, considering an aspect of Spectrum's scope that is relevant here: the work should be of high technical quality. I will likely defer to the reviewers' interpretations if the revisions fit this guideline.
## Thank you for your overall positive evaluation of our manuscript. Regarding the split opinions of the reviewers on the importance of qPCR, we would like to point out that the low abundance and limited function of lepidopteran gut microbiome has been questioned (e.g., Chen et al. 2019Chen et al. , https://doi.org/10.1002; moreover, functionally important taxa may be present in disproportionally low abundances (Xia et al. 2017(Xia et al. , https://doi.org/10.3389/fmicb.2017. Based on your suggestion on how to deal with this aspect, we chose the second option (not to do the qPCR analysis) and addressed carefully all the reviewers' points, including addition of neutral models, as suggested by the second reviewer. We also added the brief justification to the discussion, as you suggested (lines 501-507). We believe that adding neutral models gave our manuscript an additional layer of insight regarding lepidopteran microbiome assembly (especially regarding the identification of potential "core" taxa), and that our manuscript is technically sound and robust, meeting all standards of Microbiology Spectrum. More detailed responses are given below.

Reviewer #1
This study investigated seasonal shifts in the microbiomes of leaf mining larvae (Lepidoptera and Hymenoptera) and their host trees over one growing season in a deciduous temperate forest. The results not only highlight the importance of considering seasonality in the studies of the interactions between plants, herbivorous insects, and their respective microbiomes, but also clarified that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than previously presumed. This is potentially an important contribution to the insect microbiome research field; however, there are several concerns that need to be addressed.
The authors collected both Lepidoptera and Hymenoptera insects, and since the biology of the two insect orders are too different, I would suggest to separate them in the analysis and then compare the two insect orders. Furthermore, is there any interspecies differences? Whether some insect hosts have more specific gut microbiota or not.
## Thank you for this comment; the other reviewer also pointed out that confounding Hymenoptera and Lepidoptera may distort the overall message of the paper. Based on your and his/her suggestion, we decided to completely omit Hymenoptera from the analyses (there were only 21 hymenopteran samples) to avoid confounding the results, and to be able to make more meaningful comparisons with other Lepidoptera literature. The main results remained unchanged (L 105-107, 258 and onwards). We also made appropriate changes in all supplementary figures and tables. Regarding potential interspecific differences (whether some insect hosts have more specific gut microbiota or not), there weren't any differences in leaf-larval similarity among species (bacteria: df = 268, F = 1.07, P = 0.386; fungi: df = 270, χ 2 = 5.29, P = 0.871). We also analyzed differences in microbial composition among different larval species, and there were significant differences (in bacteria, host species was the variable explaining the highest portion of variability; L 282-283).
L343,"the changes were not significant", the statistical analyses (test method, P value...) should be clearly indicated in the related figure caption. Most of comparisons in the figures need be clarified.

## Done (line 793 and onwards).
High throughput sequencing is semi quantitative, representing only relative abundances. Validating some of the core taxa identified in this study (at least by Quantitative PCR) will provide more convincing evidence for the main conclusion here, because there is strong debate on the microbial residence of lepidopteran gut.
## Thank you for this comment. However, we do not think that including qPCR is necessary (and the other reviewer has the same opinion). First, we used Renkonen similarity index for the comparisons, which is independent of abundance (the patterns emerge regardless of abundance). Second, for the analysis of the taxa with seasonal trends (based on which we identified potential "core" taxa), we chose those with higher frequency in the samples to avoid making overstated conclusions based on outliers (certain taxa may be very abundant but only in a few samples). Third, in lepidopteran guts, functionally important taxa may be present in disproportionally low abundances (Xia et al. 2017(Xia et al. , https://doi.org/10.3389/fmicb.2017, which means that abundant does not necessarily equal functionally or otherwise important (abundant taxa may be only first colonizers with competitive advantage via priority effect, or are strong competitors, have their optima there, etc.). Finally, based on the suggestion of the second reviewer, we added neutral models to quantify the involvement of neutral processes in microbial community assembly, and to make our hypotheses about core taxa more supported. More detailed answers are given below.
A table summarizing the taxa prevalent in larvae, in leaves and in both leaves and larvae as well as their potential functions (in the discussion) would provide more clear information.
## Based on your suggestion, we added information about prevalence environment and potential function to Table S6. We also highlighted taxa that occurred more frequently than predicted by neutral models to make our hypotheses about core taxa more supported (in Table S6 and in the discussion, L 418-420, 430-435, 452-3, 456-8). Kindly note that there were taxa other than those highlighted in Table S6 that occurred more frequently than predicted; however, as our study aims primarily at seasonal differences, we decided to keep it uncluttered and focused on seasonal trends only.
Minors: 1. Table S5 Existing variables are less explanatory for microbial composition, and more variables should be introduced, such as temperature.
## Thank you for this comment. In our analyses, we included % irradiation as explanatory variable for both leaf and larval microbiomes. Irradiation is known to strongly affect microbial communities and is tightly linked to moisture availability and temperature stability (Šigut et al. 2022, https://doi.org/10.1038Unterseher et al. 2007Unterseher et al. , https://doi.org/10.1007Gilbert et al. 2007Gilbert et al. , https://doi.org/10.1890Gilbert et al. /05-1170Copeland et al. 2015, https://doi.org/10.1094Stone andJackson 2020, https://doi.org/10.1007/s00248-020-01564-z). The average temperature also correlates with time point that was included as explanatory variable, too. While there was a significant effect of time point on both leaf and larval microbiomes, the variable % of irradiation explained only negligible portion of variability of bacterial composition of leaves (0.78%). We also mention the effect of temperature in the discussion (lines 362-367).

Table S5
Why not distinguish the sampling sites of insects, like trees (Sampling plot)? Even if the same insect, its microbiome may also vary depending on the sampling plot (see: Foliar-feeding insects acquire microbiomes from the soil rather than the host plant).
## Sampling plot was tested as one of the explanatory variables in the analysis of larval microbiome, too. It is stated in the methods (lines 206-9). However, in Table S5, we present only variables that were significant for at least one of the components (bacteria or fungi) to keep the table uncluttered. To avoid doubts, we added this information to the caption of Table S5.